1. Technical Field
The present invention relates to a cryo transfer holder for a transmission electron microscope (which is referred to as ‘TEM’) that is configured to prevent a cooled (biological) specimen from being thermally damaged when the specimen is observed by the TEM in a cooling process.
2. Related Art
A method for observing a (biological) specimen through a TEM includes a chemical fixing method and a cryo-method. The chemical fixing method is performed by introducing a chemical fixing liquid to a fine cell structure, fixing the cell structure, cutting it to a very thin slice, and observing the cut thin slice. According to the chemical fixing method, however, the speed of the introduction of the fixing liquid to the cells is relatively slow and the cells are deformed by the chemical fixing liquid, which makes it hard to observe the cell structure in its original shape.
On the other hand, the biological specimen is basically composed of substantially low atomic weight elements. If the biological specimen is not dyed, a degree of contrast obtained by scattering is very low. By the way, since electron scattering in ice is higher than that in liquid, the water in the frozen biological specimen containing water serves to increase the contrast thereof. Because the frozen biological specimen containing water is not influenced by the chemical fixation or additional dyeing process, also, the frozen specimen is very similar to the specimen in a natural state. Therefore, the cryo-TEM method has played an important role in the field of biology.
To observe the biological specimen by the cryo-TEM method, a given procedure is needed and it is as follows: firstly, the specimen is mixed with liquid (e.g., water, buffer and the like), is placed on a given pre-processed grid by means of a pipette, and is then dewatered by means of filter paper; secondly, the specimen grid is submerged into an ethane solution cooled to a liquid nitrogen temperature and is quickly cooled so as not to form ice crystals thereon; thirdly, the quickly cooled specimen grid is transferred from a workstation insulated vessel in which liquid nitrogen is contained to a specimen cradle provided on the end of a cryo transfer holder (which is referred simply as ‘holder’). The above-mentioned processes are referred to as “specimen preparation step.” Next, the holder is mounted on a column of the TEM and an image is obtained from the specimen.
In the mounting and observing steps on the column of the TEM after the completion of the specimen preparation step, if the temperature of the mounted specimen is changed or if the temperatures between the holder and the interior of the column of the TEM are different, crystalline ice is formed on the specimen to cause the specimen tissues to be destructed, thereby influencing the quality of the observation. To stably maintain the temperature of the holder (that is, the specimen cradle) therefore, the holder typically includes a specimen rod made of good thermal conductivity and has the specimen cradle provided on one side end thereof and a thermal insulating container (Dewar) having liquid nitrogen contained therein, through which the other side end of the specimen rod is passed and insertedly fixed thereto, as shown in FIG. 1. At this time, all of the specimen rod, except the specimen cradle, is embedded into a thermal insulating pipe made of a thermal insulating material. Generally, a heater or cooler, specimen cradle rotating means and the like can be mounted around the specimen cradle in accordance with the types or purposes of the holder. Under the above-mentioned structure of the holder, the cooled air of the liquid nitrogen is effectively transferred through the specimen rod to the specimen cradle, so that the cooled specimen can be maintained well at a low temperature.
On the other hand, if the holder is mounted on the column of the TEM, the specimen rod is minutely vibrated in every direction because of its long length, and if the minute vibration is maintained, the position of the specimen cradle will be varied continuously, thereby making it impossible to perform the observation through the TEM. To prevent the uncontrollable minute vibration from occurring, a contact protrusion part, which is made of a crystal material, is attached on the end of the specimen cradle side of the specimen rod, and a contact receiving part (not shown) is provided at the position corresponding to the contact protrusion part in the interior of the column of the to be brought into contact with the contact protrusion part when the holder is mounted on the column and thus to supportingly fix the specimen rod thereto (At this time, the contact receiving part is controlled in movement by means of a goniometer of the TEM, which is not related to the present invention, and therefore, the detailed structure and movement thereof will be not explained herein). That is, if the holder is mounted on the column of the TEM, the contact protrusion part of the holder is received into the contact receiving part of the column, thereby preventing the arbitrary minute vibration from occurring.
According to the conventional practice, the cryo transfer holder for the TEM is mounted on the column (At this time, the contact protrusion part of the holder is brought into contact with the contact receiving part of the column to prevent the holder from being arbitrarily vibrated) and the pressure reduction and additional cooling of the column are performed to reach predetermined internal pressure and temperature. Through these steps, an image can be observed. However, at the moment when the cryo transfer holder for the TEM is mounted on the column to permit the contact protrusion part of the holder to be brought into contact with the contact receiving part of the tube, the heat from the tube is at a higher temperature than the holder and is thus transmitted to the specimen cradle through the contact receiving part and the contact protrusion part, thereby undesirably causing the cooled specimen to be damaged by the heat transfer.